Photodynamic Action of Methylene Blue on Pneumococcus

Abstract

In a previous study on the photodynamic action of methylene blue on bacteria 1 it was observed that such treatment exerted a lethal effect on pneumococci. Further work along this line revealed that, in addition to the lethal effect, this treatment has a preservative action on their morphology and staining property when they were kept at 37°C. Even after a period of 6 months, it was found that at the temperature mentioned, these organisms still retained their Gram positiveness comparable to fresh living cultures. On the other hand, the same organisms treated either with methylene blue alone or with formalin lost their Gram positiveness and showed signs of deterioration within a month. The procedure by which the above results were obtained is described below.

A virulent culture of pneumococcus type III grown in Avery's medium for 18 hours was mixed with a physiological saline solution saturated with methylene blue in a Petri dish in the ratio of 9 to 1. The total volume was 20 cc. The mixture was divided into 2 equal portions, one unexposed, and the other exposed to an electric light of 100 candle power at a distance of 10 cm. for a period of 45 minutes. The dish when exposed was put over a Frigidaire cooling machine which kept the temperature below 20°C. After exposure, the microorganisms were washed in physiological saline to remove the methylene blue. The samples were centrifuged and then resuspended in an equal volume of saline. They were incubated at 37°C. together with a control culture preserved with 0.4% formalin. Smears made from them were treated with alcohol for 2 hours prior to the application of Gram stain for examination giving the results stated above.